1. Field of the Invention
This invention relates to a method for preparing a cell free homogenate containing bioactive enzyme(s) and cofactor(s) of the pyrethrin pathway of Chrysanthemum cinerariaefolium (Trev.) Bocc. This cell free homogenate will convert mevalonic acid or isopentenyl pyrophospate into pyrethrins and chrysanthemyl alcohol.
2. Description of the Prior Art
Pyrethrins are a collection of six structurally related insecticidal esters found in Chrysanthemum cinerariaefolium (Trev.) Bocc. Pyrethrins I refers herein to the chrysanthemates of pyrethrin, cinerin, and jasmolin, while pyrethrins II refers herein to the pyrethrates of pyrethrin, cinerin, and jasmolin. (Cf. R. Bourne & M. Rosenthal, "(Summary of Attempts to Obtain Radioactively Labeled Pyrethrum Extract," PYRETHRUM Post, Vol. 13 (4), pp. 127-131, at 128). These compounds are effective, non-persistent insecticides used commercially in many applications, particularly in pediculocides and in preparation for insect control in industry kitchens. Pyrethrins are produced commercially by harvesting the flower heads of plants grown on farms throughout the world but particularly in Kenya and Ecuador.
Radiolabeled pyrethrins are an important tool for the evaluation and elucidation of the metabolic fate of pyrethrins in animals and insects. Absorption of labeled pyrethrin preparations through the skin or mucosal tissue of animals can assist in determining the safety of the use of such pyrethrin preparations. Labeled pyrethrins can be used to further our understanding of the fate of pyrethrins in the metabolism of target insects.
Prior art attempts to produce radiolabeled pyrethrins include the following. In John E. Casida (Ed.), PYRETHRUM--THE NATURAL INSECTICIDE (Academic Press 1973), at pages 77-79 and 106-108, several methods for radiolabeling pyrethrins are set forth. Pyrethrum plants were grown in .sup.14 CO.sub.2 or mevalonate, acetate, or methionine containing (.sup.14 C). In another method, chrysanthemic acid containing .sup.14 C was synthesized and used in turn to synthesize radiolabeled pyrethrins. In yet another method, pyrethrolone was tritiated and upon esterfication yielded tritiated pyrethrins. In Bourne & Rosenthal, supra, the above method for tritiating pyrethrolone is discussed in addition to methods for labeling pyrethrins with .sup.14 C. These methods are generally disparaged by Bourne & Rosenthal.
Cell free homogenates have been used in other fields of art to study biochemical pathways. Enzymes liberated in a cell free homogenate may be isolated and purified for subsequent use by batch or immobilized techniques as a catalyst in a desired reaction. L-amino acids are produced by the enzyme aminoacylase from Aspergillus oryzae immobilized by binding to DEAE-Sephadex. This method of producing L-amino acids is carried out by Tanabe Seiyaku Co., Japan. A cell free homogenate prepared from Gossypium hirsutum biosynthesizes gossypol from cis-cis farnesyl pyrophosphate. (P. Heinstein, et al., J. BIOL. CHEM., Vol. 245, No. 18, p. 4658 (1970)). A cell free homogenate prepared from Nicotiana tabacum tissue cultures biosynthesizes squalene from mevalonic acid. (P. Benveniste, et al., PHYTOCHEM Vol. 9, p. 1073 (1969)). A cell free homogenate from Catharanthus roseus cell suspensions synthesizes the heteroyohimbine alkaloids ajmalicine, 19-epiajmalicine and tetrahydroalstonine from geissoschizine. (Stockigt, et al., FEDS LETTERS, Vol. 70, p. 167 (1976); Stockigt, et al., PLANTA MEDICA, Vol. 37 p. 349 (1980)). U.S. Pat. Nos. 4,248,966 and 4,307,192, issued to Demain et al., issued Feb. 3, 1981 and Dec. 22, 1981 respectively, show the use of cell free homogenates for the synthesis of isopenicillin derivatives.